The invention relates to the field of biology, more specifically to the field of immunology and microbiology. The invention further relates to the field of vaccines against microbial infections and more in particular bacterial vaccines such as pneumococcal vaccines. More in particular, the invention relates to means and methods to identify, select and isolate a vaccine component to produce a vaccine for passive and/or active immunization against a microorganism that can be recognized and preferably taken up and more preferably killed by opsonophagocytic cells.
Streptococcus pneumoniae is the leading etiological agent of severe infections such as pneumonia, septicemia, meningitis and otitis media, and causes over three million deaths per year worldwide, of which a million are children. The problem of antimicrobial resistance in Streptococcus pneumoniae has been enhanced by the successful spread of a limited number of internationally recognized multi-resistant strains. These strains have complicated the treatment of pneumococcal disease, and this has further emphasized the need for vaccination as an alternative means of preventing the large-scale morbidity and mortality associated with pneumococcal infection. The initial attempt to develop an anti-pneumococcal vaccine that consisted of 23 out of 90 of the most prevalent capsular polysaccharides (Pneumovax 23©) was unsuccessful, since the vaccine polysaccharides were unable to elicit an immune response in people at most risk of pneumococcal infection: young children, immuno-compromised patients, and the elderly. Technical advances have allowed the linkage of capsular polysaccharides to highly immunogenic carrier proteins. The implementation of this technology using a vaccine consisting of tetanus toxoid linked to Haemophilus influenzae type B-polysaccharide has led to undetectable levels of carriage of Haemophilus influenzae type B, and an elimination of disease caused by this organism in vaccinated populations. Recently, a polysaccharide conjugate pneumococcal vaccine (Prevnar©) has been licensed in the USA by Wyeth Lederle Vaccines. Prevnar© has been released in The Netherlands in 2001. The vaccine formulation is immunogenic in children, and consists of 7 of the most prevalent capsule polysaccharides conjugated to a highly immunogenic (non-pneumococcal) carrier protein. However, unlike the Haemophilus influenzae vaccine, this vaccine covers only 7 out of 90 of the known pneumococcal capsule serotypes, and clinical data from phase three trials conducted in the USA, Israel, and South Africa have shown that the use of this vaccine causes a shift in colonization by non-vaccine serotypes in place of vaccine serotypes (1, 2, 3, 4, 5). As a result of this apparent weakness associated with narrow spectrum polysaccharide based antigens, attention has been focused on the non-polysaccharide capsule related pneumococcal proteins, many of which are involved in the pathogenesis of infections. These proteins are considered to be interesting components for future vaccines, since they cover all serotypes of pneumococci. The protein loci are less variable than those encoding the genes for capsular polysaccharide production, and they are more likely to be able to elicit an immune response in children (6, 7, 8, 9).
Further all the vaccines that have been developed thus far are systemic vaccines, i.e. vaccines that have to be injected. A mucosal vaccine that could e.g. be administered intranasally would be preferred for ease of administration, especially in children.
In our ongoing studies on the molecular epidemiology and pathogenesis of Streptococcus pneumoniae, we have developed a method for the extraction of surface associated pneumococcal proteins and the subsequent testing of said proteins for binding to antibodies. The antibody-antigen complex is identified, said antigen is isolated and characterized and antibodies, produced against said antigen, are subsequently tested for their opsonizing ability for the microorganism. The antigen, which is binding to an antibody with opsonizing ability for the microorganism having such an antigen on its surface, is identified by for example high-resolution two dimensional PAGE (polyacrylamide gel electrophorsesis) and mass spectrometric identification of the gel spots. This method has provided a means by which we isolated and identified a pneumococcal maturation protein (PpmA) with vaccine potential (10, 11).
The techniques used in the identification of surface-associated pneumococcal proteins, the sequence of ppmA and its protein product (PpmA) and its therapeutic potential are filed under European Patent application nr. 99202640.1.
Many microorganisms are characterized in that they are killed by a phagocytic cell after phagocytosis. Many microorganisms need to be opsonized by opsonins to enhance their phagocytosis. Opsonization is the process of making a microorganism more susceptible for the uptake by a phagocyte. In said process, opsonizing antibodies and/or proteins bind to said microorganism, thereby facilitating the uptake of said microorganism by said phagocyte. Opsonophagocytosis is measured in living phagocytic cells and comprises measuring the uptake by phagocytic cells of microorganisms pretreated with opsonizing antibodies. With this method a selection can be made for surface antigens i.e. antigens naturally or artificially associated with the surface of a microorganism that specifically induce opsonophagocytosis. Of course, surface antigens with opsonophagocytosis inducing ability can be selected from a collection of surface proteins. This typically implies that said surface proteins are isolated from microorganisms and subsequently are purified and tested.